Chronic obstructive pulmonary disease (COPD) is a debilitating disease and is the fourth most common cause of death in the US. The course of the disease is characterized by intermittent exacerbations that are associated with substantial morbidity and mortality. Nontypeable Haemophilus influenzae is the most common cause of exacerbations of COPD. Nontypeable H. influenzae can be regarded as an opportunistic human pathogen because infections occur in two selected sites in specific host settings: 1) the lower respiratory tract of adults with COPD; and 2) the middle ear of children under 6 years of age, often preceded by viral infection. Little attention has been placed on studying strains for the possibility that different strains have different virulence potential until recently. Recent studies now provide evidence that clinical isolates of H. influenzae have different combinations of genes that are associated with specific sites of human infection. Using competitive hybridization of genomic DNA from clinical isolates with a DNA microarray of genomic DNA of a COPD exacerbation strain, we have identified a pool of genes that are more likely present in COPD strains than strains from other clinical sources. These observations will be developed in the proposed studies to characterize mechanisms of pathogenesis of infection specifically in the setting of COPD. In Specific Aim 1, the distribution of genes identified by competitive hybridization that are associated with COPD will be elucidated in a large collection of well characterized clinical isolates. Further, the sequence of the genome of a prototype COPD exacerbation strain will be determined and analyzed in comparison with the genomes of previously determined otitis media strains. Bacteria alter expression of surface molecules and virulence factors in response to environmental stimuli. In Aim 2, virulence factors and surface molecules expressed under conditions simulating the human respiratory tract will be identified using an immunoproteomics approach. In Aim 3, genes identified in Aims 1 and 2 as putative virulence factors expressed during human infection will be studied to elucidate their role in pathogenesis using several models. Knockout mutants will be constructed and tested to evaluate the role of the genes in key steps in pathogenesis, including invasion of human bronchial epithelial cells, intracellular persistence, intracellular trafficking, induction of inflammation and biofilm formation. Identifying key virulence factors involved in respiratory tract infections in adults with COPD will lead to the development of fresh strategies for treatment and prevention of H. influenzae infections in COPD including, for example, novel antimicrobial agents, immunomodulators and vaccines.As the fourth most common cause of death in the US, chronic obstructive pulmonary disease (COPD) is an enormous health problem. The course of COPD is characterized by intermittent respiratory tract infections that result in hospital admissions, respiratory failure and death. This proposal includes state of the art approaches to understanding how the bacterium, Haemophilus influenzae, causes infection specifically in the setting of COPD. These results will lead directly to fresh approaches for treatment and prevention of infections by the most common cause of infection in adults with COPD.